Although chemotherapy has been responsible for curing many people of cancer, there still remain a large number of patients whose tumours either show little response to treatment, or respond initially only to recur later. For these patients the current treatments are clearly inadequate.
It is thought that certain tumours are unresponsive to conventional chemotherapy because the cells of these tumours have a pattern of gene expression that renders them insensitive to chemotherapeutic agents. Similarly, it is thought that tumours often respond initially to chemotherapy, but subsequently become resistant because the cells of the tumour exhibit tumour heterogeneity and genetic instability. Tumour heterogeneity describes the situation where different cells in the tumour have different patterns of gene expression with some cells being resistant to a chemotherapeutic agent, whilst other cells are sensitive to this agent. Treating such a tumour with this chemotherapeutic agent therefore kills the sensitive cells, resulting in tumour shrinkage, but fails to kill the resistant cells, which continue dividing to produce a cancer that is wholly drug resistant.
In addition most conventional chemotherapeutic agents developed up to the present time generally inhibit the growth of important normal cells, for example: a) chemotherapeutic inhibition of the progenitor cells of the haemopoietic system resulting in a fall of red blood cells, white blood cells and platelets causing anaemia, susceptibility to infection and spontaneous bleeding b) inhibition of replacement of normal cells in the bowel causing diarrhea or c) inhibition of replacement of squamous cells lining the mouth, nose and throat etc.
Genetic instability is found in the majority of cancers. It results in the tumour cells acquiring new mutations. Certain of these mutations may confer drug resistance to the cells in which they occur. These drug resistant cells survive chemotherapy and divide to produce a cancer that is drug resistant.
There is thus a need for anticancer agents which are effective against all cancer cells, which are not affected by tumour heterogeneity and genetic instability and which do not inhibit growth of normal (non-cancerous) cells or which may even promote normal non-cancerous cell growth.
WO 03/081239, which is hereby incorporated in its entirety by reference, identifies gene products, termed critical normal gene products, which are required for cancer cell survival and proliferation. Because critical normal gene products are required for cancer cell survival and proliferation, they must be present and functioning in every tumour cell and therefore provide a consistent anti-cancer drug target that is unaffected by tumour heterogeneity and genetic instability. WO 03/081239 teaches that agents that disrupt critical normal gene products provide effective anti-cancer agents. Although generic methods for disrupting critical normal gene products were disclosed, WO 03/081239 did not disclose any agent that could successfully treat cancer.
Critical normal gene products should also, by definition, not disrupt the function of normal cells. Thus, conventional chemotherapy in the clinic is non-selective and thus consistently damages normal non-cancerous cells and is only effective against non-resistant cancer cells.
An ideal anticancer agent would inhibit the growth of most, if not all, types of cancer cell growth but have no effect on, or even stimulate, normal non-cancerous cell growth.
WO 03/081239 identified CDK4 protein as a critical normal gene product that is present in most (if not all) cancers.
CDK4 protein is known to regulate entry into S phase of the cell cycle by initiating the events needed for the cell to enter S phase. More particularly, activated CDK4 phosphorylates pRb and related proteins p107 and p130. In their hypophosphorylated state these proteins bind E2F transcription factors. However, upon phosphorylation, the E2F transcription factors are released as heterodimers with the proteins DP-1/DP-2. The E2F/DP heterodimers then bind to DNA and activate factors required for DNA synthesis (an activity that takes place during S phase). In addition, free E2F protein upregulates genes controlling cell division such as cyclin E, cyclin A, CDK1 and E2Fs, thereby progressing the cell cycle.
CDK4 protein is only activated when conditions for entry into S phase are suitable and positive signal transduction pathways relaying signals from cell surface receptors such as the Ras/Raf/Erk pathway have been demonstrated to affect CDK4 activation. CDK4 protein is activated by phosphorylation of threonine 164 but inhibited by phosphorylation of tyrosine 17.
To enable it to perform its role, CDK4 protein is known to have many functions including binding cyclin D1, phosphorylating pRb, binding to CDK inhibitors such as p21, p27, p16, binding to cyclin activating kinase and interacting with the enzymes responsible for phosphorylating and dephosphorylating tyrosine 17.
Because of its role in promoting cell division, several studies have investigated the role of CDK4 protein in cancer.
Knockout mice lacking CDK4 protein do not develop cancer following induction with a classical system of initiator (DMBA) followed by promoter (TBA i.e. phorbol ester) (Robles et al. (1998) Genes Dev. 12: 2469; Rodriguez-Puebla et al. (2002) Am. J. Path. 161: 405). No other knockout (including a cyclin D1 knockout) has such a marked effect on cancer development.
However, the CDK4 protein is typically over-expressed in cancer cells. In addition, transgenic mice overexpressing CDK4 protein are more readily induced to develop cancer using the carcinogenesis induction system mentioned above (Robles et al. (1998) Genes Dev. 12: 2469; Rodriguez-Puebla et al. (2002) Am. J. Path. 161: 405).
Moreover, transfection of normal CDK4 has been shown to cause extension of proliferative lifespan in normal human fibroblasts (Morris et al. (2002) Oncogene 21, 4277)
In view of the apparent importance of CDK4 protein in cancer, it has been proposed to be an anticancer target. However, drugs that inhibit CDK4 kinase activity (such as flavopiridol) have very little clinical effect in phase II studies.